Perturbations in skeletal muscle mitochondrial (mt) protein expression may contribute to the pathogenesis of metabolic disorders in HIV-infected people treated with thymidine-analog-based antiretroviral therapy (AZT- or d4T-ARV). Skeletal muscle is the largest, mt-rich tissue in the body and the primary site for glucose storage. Normal muscle mt protein expression is required for normal glucose and fatty acid metabolism. However, we lack sensitive, specific and comprehensive analytical tools for examining the human muscle mt proteome. We propose to develop sensitive, mt-specific, mass spectrometry-based comparative proteomics tools and approaches that can be used to identify, characterize, and quantify the human muscle mt proteome in specimens previously obtained from 4 groups of well characterized subjects: HIV-seronegative with normal glucose tolerance (NGT); HIV+ naive to ARV with NGT, HIV+ receiving AZT- or d4T-based ARV with NGT; and HIV+ receiving AZT- or d4T-based ARV with insulin resistance. We hypothesize that AZT- or d4T- based regimens impair muscle mt protein expression and induce post-translational modifications to mt proteins that are associated with HIV-related insulin resistance. Specifically, we will separate muscle mt proteins using customized sub-cellular fractionation, protein enrichment and depletion methods, 2D- differential fluorescence gel electrophoresis, and 1D-liquid chromatography. We will identify and characterize muscle protein/peptides using a variety of mass spectrometers (MALDI-TOF, LC-ESI-, nano-LC-FT-tandem MS) for accurate mass measurements and amino acid sequencing. We will discover new and important mt protein forms that will generate novel approaches and new hypotheses about the pathogenesis of muscle mt-based metabolic disorders in HIV-infected people. These analytical approaches and tools have been used to examine proteomes in small organisms, but we need to advance and facilitate their application to complex human tissues (muscle) that are critically involved in disorders of human substrate metabolism, and that might ultimately lead to novel treatment strategies. To accomplish this, we will take advantage of the expertise and the wide variety of high resolution mass spectrometry instrumentation available at Washington University.